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Muscle biopsy interpretation
1. DR. NOOPUR S. PATIL
JR2,DEPT OF PATHOLOGY
MIMSR MEDICAL COLLEGE,LATUR
INTERPRETATION OF
MUSCLE BIOPSY
2. INTRODUCTION
Microscopic examination of muscle provides
useful information for diagnosis, prognosis and
treatment of muscle diseases
Duchenne in 1868 introduced muscle biopsy
Evaluation of skeletal muscle biopsy needs to be
done in context of clinical history, examination of
patient and other tests including serum CK and
EMG.
3. INDICATIONS
A) GENERAL
Weakness of uncertain cause – generalised,
proximal, floppy infant syndrome
Muscle pain, cramps, stiffness
Persistently elevated muscle enzymes
B) SPECIFIC
Hereditary muscle disease in other family members
Carrier detection
Systemic connective tissue disease and vasculitis
4. Storage diseases
Suspicion of steroid myopathy in treated myositis
To exclude drug induced myopathy
Confirm/reinforce clinical diagnosis
Conflicting clinical, EMG, laboratory findings
5. CONDITIONS IN WHICH MUSCLE BIOPSY IS LOW YIELD
OR CONTRAINDICATED PROCEDURE
Electrolyte disturbances
Most endocrine diseases
Malignant hyperthermia
Myasthenic syndromes
Myotonic disorders
Old age
Poor nutrition
6. Choose a moderately affected muscle MRC grade 3,4
Medical research council grading of muscle power
Grade 0 – no movement
Grade 1 – flicker of contraction
Grade 2 – movement with gravity eliminated
Grade 3 – movement against gravity
Grade 4 – movement against moderate resistance
Grade 5 – movement against full resistance( full power)
7. COLLECTION AND PREPARATION OF MUSCLE
BIOPSY SPECIMEN
Open biopsy/needle biopsy
Advantages of needle biopsy
1) outpatient procedure
2) use of local anaesthesia
3) minimal scarring
4) ability to sample multiple sites
Disadvantages of needle biopsy
1) limited sampling
8. • Specimen should be
representative of disease
obtained from a muscle in which disease is active
and evolving
taken from the belly of muscle
• Muscles subjected to prior trauma or affected by
unrelated disease – not sampled
• Two separate specimens are routinely requested
Before excision first specimen is maintained in
isometric state by introducing it in muscle clamp
9. preventing contraction artifact. Acceptable sample
size is 1 x 0.5 cm. The fixed sample is used for
paraffin sections, 1-2 micron resin embedded
sections, EM studies
Second fresh specimen measuring 1x0.5x0.5 cm is
used for frozen sections preparation,
immunofluorescence microscopy/biochemical
analysis along with H & E, rapid Gomori Trichrome,
ATPase, NADH-TR.
Once specimen is obtained it should be transported
in saline moistened gauze in timely fashion
10.
11. • The technique is safe and highly effective using a modified Bergström needle to
obtain skeletal muscle tissue samples from the vastus lateralis of human
subjects.
• The Bergström needle consists of an outer cannula with a small opening
('window') at the side of the tip and an inner trocar with a cutting blade at the
distal end.
• Under local anesthesia and aseptic conditions, the needle is advanced into the
skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia.
• Next, suction is applied to the inner trocar, the outer trocar is pulled back,
skeletal muscle tissue is drawn into the window of the outer cannula by the
suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal
muscle tissue sample.
• The needle is rotated 90° and another cut is made.
• This process may be repeated three more times.
• This multiple cutting technique typically produces a sample of 100-200 mg or
more in healthy subjects and can be done immediately before, during, and after a
bout of exercise or other intervention.
13. Weil-Blakelsley Cochotome
Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in
both clinical and research practice. It is a safe, effective and well tolerated technique.
The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is
inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue
penetration. The tip is opened and closed within the tissue and then rotated through 90 -
180° to cut the muscle. The amount of muscle obtained following repeated sampling can
vary from 20 mg to 290 mg which can be processed for both histology and molecular
studies.
17. INTERPRETATION OF MUSCLE BIOPSY
SPECIMEN
1) Normal muscle
Fiber types are not evident by H & E
Myofibrillar ATPase is considered to be most
reliable method for distinguishing fiber types.
18. Transverse section of muscle fibres, typically polygonal, and
the sarcolemmal nuclei are located peripherally
19.
20.
21. HISTOLOGY
Skeletal muscle is composed of extremely elongated,
multinucleate contractile cells(muscle fibers) bound
together by collagenous supporting tissue.
Contraction is controlled by large motor
nerves,individual nerve fiber branches within the muscle
to supply a group of muscle fibers called motor unit.
Every muscle fiber is a syncytium formed as a result of
fusion of several hundreds of component myoblasts
Individual muscle fiber diameter 10-100 µm
Adult muscle fibres on transverse section are polygonal
(round in infancy)
Each muscle fibre is composed of numerous myofibrils
and myocyte cytoplasm(sarcoplasm)
22. Each myocyte is multinucleated, nuclei are subsarcolemal
in location
Normally only 3-5% of myocytes will have internally
placed nuclei
Each myofibril consists of identical repeating
units(sarcomeres) composed of myofilaments (Actin and
Myosin)
Arrangement of contractile proteins gives rise to
appearance of cross-striations
Myofibril interacts with sarcolemma through cytoskeletal
protein dystrophin
Epimyesium, perimyesium, endomyesium (connective
tissue sheath)
Endomysium surrounds individual muscle fiber
Perimysium envelopes groups of muscle fiber i.e fascicle
Epimysium envelops many fascicles i.e. whole muscle
24. Type - 1 Type -2
Red /oxidative/slow glycolytic/fast/white
* ATPase Low High
*Oxidative enzyme High Low
*Glycogen Low High
*Phosphorylase Low High
*Lipid content High Low
Inc. Myoglobin. Dec myoglobin
Function Postural activity Sudden
intermittent activity
Metabolism Aerobic Anareobic
35- 40% 60-65%
TYPES OF MUSCLE FIBERS
29. NUCLEAR CHANGES
• Normal nuclei- peripheral
• Increase in internal nuclei –M/C abnormality
• Large no. of myofibers with central / paracentral
nuclei - S/o Myopathic diseases.
30. Internal nuclei are seen in:-
Myotendinous insertion
Fiber atrophy - multiple pyknotic nuclei forming
clusters.
Fiber regeneration – vesicular nuclei with
prominent nucleoli.
Centronuclear myopathy- Single central/
paracentral nucleus in almost all fibers-
Myotonic dystrophy- randomly distributed nuclei
with active appearing nuclei (dispersed chromatin)
31. RING FIBERS
F
• Formed by peripheral bundle of myofibril.
• Directed circumferentially,
encircling inner portion of myofiber.
• Seen in transverse section.
• Striation are visible under PAS,
Resin & EM
• Normally seen in extraocular muscles.
• Seen in Limb girdle dystrophy & Myotonic dystrophy
• Large no. of ring fibers – S/O Myotonic dystrophy.
32. HYALINE FIBERS ( DEGENERATION)
Degenerating round and enlarged
More deeply stained than normal
Sarcoplasm – smudged, homogenous
Nuclei – Pyknotic in centre / periphery
In DMD- Large no. of Hyaline fibers
35. Loss of striation
Swelling of myofiber eosinophilia
(acute necrotic fibers)
Later pale color
Sarcoplasm striated to coarsely granular
Nucleus –Pyknotic,fragmented ,absent
Macrophage seen in surrounding
fibers
FIBER NECROSIS
Presence of degeneration & necrosis – definite sign of myopathy
Best seen in H&E section
Myophagocytosis
36.
37. Pathological features Disease
Small groups of necrotic fibers Duchenne dystrophy
Perifascicular necrosis Dermatomyositis
Random fiber necrosis Polymyositis, inclusion body myositis
Infarcts with large areas of necrosis Polyarteritis nodosa
Extensive, diffuse necrosis Rhabdomyolysis in alcoholics, military
recruits
38. FIBER REGENERATION
Degeneration/ Necrosis
Compensatory regeneration
Increased basophilia
Source of regeneration:
1.Sprouts of remaining
sarcoplasm
2.Satellite cells – more
capacity to regenerate.
Regenerating fibers – increased basophilia
Nuclei in number, larger than normal
With vesicular chromatin, prominent
nucleoli.
39.
40.
41. Presence of regenerating fibers even in
absence of necrotic fibers is indicator of
previous necrosis or fiber necrosis of
adjacent muscle
Satellite cells with restorative capacity play
important role in regeneration
42. FIBER SPLITTING
Hypertrophic fibers split into 2/>2 subunits.
A split like space from invagination individual
segment.
Limb girdle dystrophy
Inclusion body myositis
Mechanism- (A form of
Regeneration)-failure
to unite to form a
single fiber
45. Interstitial inflammatory infiltrates most frequently
encountered in polymyositis, dermatomyositis,
inclusion body myositis
Nodular infiltrates composed largely of plasma cells
highly suggestive of rheumatoid arthritis
PAN and SLE associated with vasculitis
Granulomatous inflammation indicative of
sarcoidosis or idiopathic granulomatous myositis
46. Fibrosis and fatty infiltration
Bequest of chronic neuromuscular disease of both
myopathic and neurogenic origin
47. ATROPHY
MC histological change
Interpretation better in cross section & in frozen
section.
General causes of
atrophy( non
selective )-
1. Denervation- MC
2. Disuse
3. Ischemia
4. Aging
5. Poor nutrition.
49. MORPHOMETRIC ANALYSIS OF ATROPHY
Done either manually / computer assisted image
analyzer.
At least 200 fibers should be present in the
sample.
Interpretation:
Grouped atrophy- 5/>5 angular fibers –
pathognomonic for chronic neurogenic disease
Panfascicular atrophy – Infantile spinomuscluar
atrophy
Perifascicular atropy – DM
Atrophic fibers randomly situated in the section
is non specific.
50. FIBER HYPERTROPHY
Type 1:
ISMA
Type 2:
Runners sprinters
Congenital fiber type
disproportion
• Limb – girdle
dystrophy
• IBM
• Myotonia
congenita
• Acromegaly
51. Type-1 Type-2A Type 2B
1)ATPase reaction Light Dark Intermediate
at 9.4 pH
2)ATPase reaction Dark Light Intermediate
At acidic pH
3)NADH-TR Reac.
Tetrazolium reductase Dark Intermediate Light
Depend on conc.of
mitochondria
4)Antibodies
Slow +nt -nt -nt
Fast - nt +nt +nt
DIFF. BETWEEN MUSCLE TYPES DONE BY
HISTOCHEMICAL STAIN ON FROZEN
SECTION
52. ATPase stain pH 9.4
On ATPase stain we can see normal checkboard pattern
of intermingled type 1 and type 2 fibers
54. 2) Modified gomori trichrome
Highlights ragged red fibers, rods, rimmed vacuoles
3) PAS – glycogen
4) Oil red O/Sudan black - lipid
5) NADH-TR
Less specific than ATPase
Type I darker than type II
Useful stain in diagnosis of mitochondrial myopathy
and structural myopathy (central core disease)
In denervation target and targetoid fibers are also
best demonstrated by this stain
56. 6) Succinic dehydrogenase
Type I darker than type II
Ragged red fibers highlighted by SDH
7) Acid phosphatase
• Highlights lysosomal associated abnormalities such
as acid maltase deficiency
61. CORE & TARGETS
Better seen in oxidative enzyme stain
Targets
Appear as central pallor s/by darkly stained rim, in turn s/by normal
appearing area.
Cores
Appear as a central pallor s/by normal appearing area without dark
zone.
Target Cores
62. Target Cores
Always single Single/multiple &
eccentric
Extends upto few sarcomere Extends throughout
the length
> diameter. < diameter
Pathognomonic for Neurogenic Central core disease
atrophy
68. Vacuolar degeneration
1. Dilatation of the sarcotubular system
Vacoules may be empty or may contain PAS +ve staining
material
e.g hypo and hyperkalaemic periodic paralysis
2. Autophagic vacoulation – represent membrane bound
areas of autodigestion of muscle fibre
e.g mytonic dystrophy, polymyositis, drug induced
myopathy due to chloroquine and vincristine
3. storage vacoules – excessive accumulation of neutral
lipids and glycogen
69. Immunohistochemistry
Sarcolemma-related proteins –Dystrophin.
Absent dystrophin:
Has specificity for Duchenne Muscular Dystrophy
Reduced dystrophin:
Patchy staining of sarcolemma of individual fibers
may occurs in Becker Muscular dystrophy
81. Muscle Dystrophy
Heterogenous group of inherited disorders beginning in
childhood
Clinically – progressive muscle weakness and wasting
Duchenne’s muscular dystrophy : X-linked
Becker’s muscular dystrophy : X-linked
Limb girdle muscle dystrophy : AD/AR
Occulopharyngeal muscle dystrophy : AD
Facioscapulohumeral muscle dystrophy : AD
Emery-Dreifuss muscular dystrophy : X-linked
Congenital muscle dystrophies : AR
82. Duchenne muscular dystrophy
1 per 3500 live births. Most common muscular dystrophy
Manifest by 5 years of age
Boys frequently fall, difficulty in running, jumping
On getting up from floor child uses his hands to climb up
(Gower’s sign), contracture of heel cords and ileotibial
bands
By age of 12 child becomes wheel chair dependent,
scoliosis
Predisposed to lung infections, aspiration of food, acute
gastric dilation, cardiomyopathy
Lab findings : CK levels 20-100 times the normal, late in
course of disease the levels decrease due to inactivity and
loss of muscle mass
EMG : features typical of myopathy
83.
84. Pathogenesis : Deletion affecting gene Xp21 that encodes
dystrophin, complete absence of dystrophin
Dystrophin and dystrophin associated protein complex
form an interface b/w intracellular contractile apparatus
and extracellular connective tissue matrix
Dystrophin is located adjacent to sarcolemmal membrane
in myocytes
85. Microscopy : Both type1 and type 2 fibers are involved
Variation in fiber size : presence of both small and large
fibers, fiber splitting
Increased numbers of internalized nuclei(>3-5%)
Enlarged rounded hyaline fibres that have lost their
normal cross striations – hypercontracted fibres
Degeneration, necrosis and myophagocytosis
Regeneration of muscle fibers
Proliferation of endomysial connective tissue
In later stages muscle eventually becomes almost totally
replaced by fat and connective tissue.
Cardiac involvement : interstitial fibrosis in
subendocardial layers
IHC : lack of dystrophin in DMD and decreased amount
in BMD
91. Becker’s muscular dystrophy
X-linked recessive condition
Defect in the dystrophin gene – structural alteration or
decrease in size of the molecule
Onset later as compared to DMD
Child can walk past 15 years of age and survive till
adulthood
Microscopy – Variability of fiber size, scattered large
hypercontracted fibres, endomysial fibrosis. Fiber necrosis
and regenerative changes are much less conspicuous than
Duchenne muscular dystrophy
92. Facioscapulohumeral muscle dystrophy
7-27 years of age
Associated with mental retardation and epilepsy
Initial weakness is usually seen in facial muscles in
particular the zygomaticus, orbicularis oculi, orbicularis
oris. Masseter, temporalis and extraoccular muscle are
spared. Weakness of scapular muscle i.e latismus dorsi,
lower trapezius, rhomboids, serratus anterior
M/S : Myopathic changes
variation in fiber size, increase frequency of central
nuclei, occasional necrotic fiber, increase in endomysial
and perimysial connective tissue.
Lobulated fibers may be seen
99. Occulopharyngeal muscle dystrophy
Late onset 5th-6th decade, AD
Two cardinal symptoms : Ptosis, dysphagia
All muscle are affected but extraoccular, lingual,
pharyngeal and diaphragmatic muscle are selectively
more involved.
M/S : i. loss of muscle fiber
ii. Variation in fiber size,
iii.increase in number of internal nuclei ,
iv. Increased interstitial and fibrous connective tissue,
v. small angulated fiber,
vi. rimmed vacuole within muscle fiber.
vacuoles lined by a ring of material which appears
basophilic on H & E stain and red on Gomori’s trichrome
100. Myotonic dystrophy
AD disease, increased CTG repeats at chromosome 19
Clinically two forms : congenital & adult form
Congenital form : infants, hypotonia and myotonia
Adult form : slow onset with progressive weakness and
stiffness of the distal limbs. Muscle of the face, jaw and
eyelids are most frequently involved. Myotonia results in
marked delay in grip release.
Male pattern of baldness, cataracts, testicular atrophy,
mental subnormality, cardiac arrythmias, cardiomyopathy
101. M/S : type 1 fiber atrophy, type 2 fiber hypertrophy,
increase internal nuclei which in longitudnal section
forms conspicuous chains.
Ring fibers – subsarcolemmal band of cytoplasm that
appears distinct from the center of the fiber. The rim
contains myofibrils that are oriented circumferentially
around the longitudinally oriented fibrils in rest of fiber
Sarcoplasmic mass – ring fiber may be associated
with an irregular mass of sarcoplasm, extending outward
from the ring. These stain blue with H&E, red with MGT,
intensely blue with NADH-TR histochemical reaction
Of all the dystrophies only myotonic dystrophy shows
pathologic changes in the intrafusal fibers of muscle
spindles, with fiber splitting, necrosis and regeneration
102. Ring fibers. Circumferentially oriented myofibers are
seen at the periphery of transversally sectioned fibers.
(Cryosection, modified Gomori trichrome).
103.
104. Emery-Dreifuss muscular dystrophy
X-linked , mutation in STA gene which encodes emerin,
inner nuclear membrane protein
Muscle biospsy : variation in fiber size with abundant
small fibers, increase internal nuclei, mild regeneration,
some necrotic fibers, type 1 fiber predominance or type 2
predominance and type grouping .
IHC : absence of Emerin
106. CENTRAL CORE DISEASE
AD
Chr.19q13.1
Affects RYR1gene – inv. Ryanodine receptor prot.
Type 1 fibers are affected
Mild , proximal, non progressive muscle
weakness.
Cores are seen as regions of depleted or absent
oxidative enzyme activity.
Biopsy –
1. Many fibers show single centrally located defect or core.
2. More than one core per fiber may be encountered.
108. MULTI CORE DISEASE
Congenital, non progressive myopathy
Known as Minicore disease
Generalised weakness and hypotonia.
Kyphosis, scoliosis and muscle contractures
Type 1 fiber predominance.
Biopsy-
Numerous , multiple core like structure in majority
of muscle fibers.
PAS/ Trichrome/ NADH-TR – stains pale
110. Nemaline rod myopathy
Originally described by Shy et al. and canon et al in 1963
Hypotonia, floppy infant, non-progressive myopathy
Rods are composed of actin and myosin
Rods are not visible on H&E or oxidative enzyme stains
Best demonstrated on MGT stain – subsarcolemmal dark
red rods, measure 3-6 µ in length and 1-3 µ in width
Exclusively seen in type 1 fibres, there is usually type 1 fiber
predominance
EM – subsarcolemmal elongated or rectagular rods in
longitudnal section, polygonal in cross section
114. Centronuclear myopathy
Myotubular myopathy, X-linked , AD, AR
Slowly progressive or non-progressive hypotonia, proximal
or generalized weakness
External ophthalmoplegia and facial weakness
Microscopy : Many fibres with central nuclei more in type 1
fibers, type 1 fiber predominance and type 1 fiber atrophy,
central perinuclear clear zones may stain intensely with
PAS and oxidative enzymes (SDH)
On longitudinal section nuclei appear to be in a single row
(rowing of nuclei)
Radiating spoke pattern on NADH-TR stain
115. Most fibers display central nucleus. Note a perinuclear
halo of abnormality of myofibrils. (Cryosection, H&E).
116. Congenital fiber type disproportion
AD, AR or Sporadic
Hypotonia , associated with joint contractures, Congenital
dislocation of hip, Skeletal deformities
In ATPase stain (pH 9.6) type I fibers are 12% or more
smaller in size than type II fibers.
123. Perivascular inflammation, focally in perimysium
and less commonly in endomysial compartment
Vasculopathic – endothelial hyperplasia, necrotising
vasculitis, fibrin thrombi, obliteration of small
vessels
128. Endomysial chronic inflammatory infiltrate similar
to polymyositis
Fiber hypertrophy and splitting, rare in other
inflammatory myopathies are typical features
Small group atrophy of fibers mimics neurogenic
atrophy
Most characteristic feature ultrastructurally is
presence of intranuclear, cytoplasmic
tubulofilaments
129. HIV myositis
Closely resembles polymyositis pathologically
Nemaline rods in some patients
Multinucleated giant cells similar to that described
in brains of AIDS patients
140. SUMMARY
• A skeletal muscle biopsy is important for the
diagnosis of diseases of motor unit, systemic diseases
such as vasculitis and disorders of metabolism such
as glycogenosis.
When interpreting the biopsy, the pathologist must
have knowledge of patient’s clinical and family
history, physical examination findings, results of
EMG, nerve conduction and serum creatinine
phosphokinase.
141. Light microscopic characteristics to be noted:
1) Size and shape of myofiber
2) Position of nuclei
3) Hyalinisation, necrosis, myophagocytosis
4) Vacuolar changes
5) Presence of intracytoplasmic inclusion bodies
6) Ring fibers
7) Regenerative activity
8) Increased endomysial and perimysial connective
tissue
142. Histochemical features to be noted:
1) Fiber type distribution in fascicles
2) Involvement of particular fiber type
3) Predominance of any one fiber type
4) Grouping of fibers of one histochemical type
5) Abnormal mitochondria, cores, vacuoles, targets
6) Enzyme deficiencies can also be identified
143. At ultrastructural level changes in sarcolemma,
myofilaments, mitochondria, Z bands have to be
documented.
144. Commonly encountered muscle diseases like
Duchenne dystrophy, denervation atrophy and
inflammatory myopathies can be diagnosed by
routine histology.
Metabolic myopathies, congenital myopathies need
additional procedures for identification
Introduction of enzyme histochemistry and IHC has
revolutionalised the diagnostic efficacy in
myopathology enabling initiation of appropriate
treatment to patient and counselling to patient’s
relatives
145. Though the procedures of muscle biopsies and the
laboratory techniques are elaborate and time
consuming, they are essential for diagnosis.
146. REFERENCES
1) Heffner RR Jr, Balos LL. Muscle Biopsy in
Neuromuscular Diseases. In: Mills, Carter,
Greenson, Oberman, Reuter, Stoler (eds.)
Sternberg’s Diagnostic Surgical Pathology. Vol.1.
4th ed. USA. Lippincott Williams and Wilkins;
2004. p111-36.
2) Ang LC. Skeletal Muscle. In: Rosai J (ed.) Rosai and
Ackerman’s Surgical Pathology. Vol.2. 9th ed. India.
Elsevier; 2009. p2663-82.
147. 3) Prayson RA. Muscle and Peripheral Nerve
Pathology. In: Silverberg, Delellis, Frable, Livolsi,
Wick (eds.) Silverberg’s Principles and Practice of
Surgical Pathology and Cytopathology. Vol.2. 4th ed.
China. Churchill Livingstone; 2006. p2213-66.
4) Heffner RR Jr, Schochet SS Jr. Skeletal Muscle. In:
Damjanov I, Linder J (eds.) Anderson’s Pathology.
Vol.2. 10th ed. USA. Mosby-Year Book, Inc.; 1996.
p2653-92.
148. 5) Anthony DC, Frosch MP, Girolami UD. Peripheral
Nerve and Skeletal Muscle. In: Kumar, Abbas,
Fausto, Aster (eds.) Robbins and Cotran Pathologic
Basis of Disease. 8th ed. Pennsylvania. Elsevier;
2010. p1257-78.
6) Workshop on Muscle Disorders conducted by
Department of Pathology, J.N. Medical College,
Belgaum on 12th June 1993.